I. Muscle Cells
The three muscle cell types, skeletal, cardiac, and smooth, are each derived from distinct populations of myogenic precursor cells during embryogenesis (Christ et al, Anat. Embryol., 191:381-396 (1995); and Schwartz et al, Physiol. Rev., 70:1177-1209 (1990)).
Skeletal muscle arises from the somites, which form adjacent to the neural tube beginning at about embryonic day 8 (E8) in mice. Subsequent compartmentalization of the somites gives rise to the myotome, from which the axial musculature is derived (Buckingham, Trends Genet., 8:144-148 (1992)). Cells from the ventrolateral edge of the dermamyotome of the somite also migrate into the limb buds to form the limb muscles (Buckingham, supra).
Cardiac muscle is derived from the anterior lateral plate mesoderm, which forms a primitive heart tube at about E8, and subsequently undergoes looping and chamber specification to form the mature multi-chambered heart (Kaufman, The Atlas of Mouse Development, Academic Press, San Diego (1992)).
The embryonic origins of smooth muscle cells (SMCS) are less clear, in part because they arise in multiple regions of the embryo from different precursor populations. For example, studies in chick/quail chimeras have shown that SMCs in the great vessels are derived from a subpopulation of mesenchymal neural crest cells, whereas SMCs in the coronary arteries are of non-neural crest origin (Hood et al, Anat. Rec., 234:291-300 (1992); Rosenquist et al, Ann. NY Acad. Sci., 588:106-119 (1990); Kirby et al, Science, 220:1059-1061 (1983); and Le Lievre et al, J. Embryol. Exp. Morphol. 34:125-154 (1975)).
In addition to vascular SMC, there exists several seemingly distinct populations of SMC in most visceral organs, i.e., those of the respiratory, gastrointestinal and genito-urinary systems. The visceral SMCs are thought to originate from local mesenchyme, apparently through inductive processes (Cunha et al, Epith. Cell Biol., 1:76-83 (1992)).
II. Properties of Smooth Muscle Cells
SMCs are important for the functions of the circulatory, genito-urinary, respiratory and digestive systems. Unlike skeletal and cardiac muscle cells, where cell differentiation is accompanied by stable expression of muscle-specific genes (Weintraub et al, Science, 251:761-766 (1991); and Olson, Genes Dev., 4:1454-1461 (1992)), SMCs display remarkable phenotypic plasticity, and retain the capacity to re-enter the cell cycle (Schwartz et al, Circ. Res., 58:427-444 (1986)). This unique property of SMC phenotypic modulation is often associated with the loss of many SMC-specific markers (Glukhova et al, Am. J. Physiol., 261:78-80 (1991); and Frid et al, Dev. Biol., 153:185-193 (1992)). Such alterations in SMC proliferation and differentiation are associated with a variety of vascular diseases including atherosclerosis, restenosis following angioplasty, and hypertension (Schwartz et al (1986), supra; and Glukhova et al, supra).
From the above, it is clear that smooth muscle shows unique properties in terms of both differentiation control and ontogeny when compared to sarcomeric muscle.
In contrast to skeletal and cardiac muscle cells, where the molecular mechanisms governing muscle-specific gene expression are beginning to be understood (Olson, Circ. Res., 72:1-6 (1993)), relatively little is known about the mechanisms controlling muscle gene expression in SMCs. Further, relatively little is known about the molecular mechanisms that control the SMC myogenic program, which are believed to be important for developing therapeutic strategies for the treatment of human vascular diseases.
Only a few SMC-specific marker genes, i.e., that are expressed specifically in adult SMCs, have been studied extensively in vitro with respect to transcriptional regulation. However, no in vivo studies have been carried out. Among these genes are smooth muscle (SM) .alpha.-actin (Foster et al, J. Biol. Chem., 267:11995-12003 (1992); and Shimizu et al, J. Biol. Chem., 270:7631-7643 (1995)), and smooth muscle myosin heavy chain (SM-MHC) (Katoh et al, J. Biol. Chem., 269:30538-30545 (1994)). Another gene, SM22.alpha. has been less well characterized (Duband et al, Differentiation, 55:1-11 (1993); Nishida et al, Gene, 130:297-302 (1993); Shanahan et al, Circ. Res., 73:193-204 (1993)); and Lees-Miller et al, Biochem. J., 244:705-709 (1987)).
III. SM22.alpha.
SM22.alpha. is considered to be a SMC-specific protein structurally related to calponin, which is an actin- and tropomyosin-binding protein (Winder et al, Adv. Exp. Med. Biol., 304:37-51 (1991)). SM22.alpha. also shows homology to the Drosophila protein mp20, which is expressed specifically in synchronous oscillatory flight muscles, but not in asynchronous flight muscles (Ayme-Southgate et al, J. Cell Biol., 108:521-531 (1989)). In addition, SM22.alpha. shows homology to protein NP25, which is expressed specifically in a subpopulation of neuronal cells (Ren et al, Mol. Brain Res., 22:173-185 (1994)).
There are three isoforms of SM22.alpha., but the .alpha.-isoform is the most abundant one (Lees-Miller et al, supra; and Lees-Miller et al, J. Biol. Chem., 262:2988-2993 (1987)).
SM22.alpha. has been shown to be expressed in all smooth muscle tissues of birds and mammals. However, SM22.alpha. mRNA expression during embryogenesis has not been examined. Thus, to begin to define the mechanisms that control muscle gene expression during SMC differentiation, and to further characterize the embryonic origins of SMC lineages, the murine SM22.alpha. gene was cloned in the present invention, and its mRNA expression pattern examined during murine embryogenesis.
IV. Targeted Gene Therapy
Several reports have described the use of adenoviral or liposome-mediated gene transfer in the vessel wall to potentially treat vascular diseases that account for more annual deaths than all cancers combined. These reports have relied on constitutively active DNA viral promoters or liposomes to direct expression of foreign DNA in SMCs (Nabel et al, Science, 249:1258-1288 (1990); Stewart et al, Hum. Gene Therapy, 3:267-275 (1992); Nabel et al, Nature, 362:844-846 (1993); and Chang et al, Science, 267:518-522 (1995)). Despite successful transfer of a number of recombinant proteins that can influence the biology of SMC in vivo, these previously known gene transfer vectors do not assure SMC specificity. That is, the transfected DNA is taken up by other vascular cells, especially the vessel lining endothelial cells.
Accordingly, there has been a desire in the art isolate SMC-specific promoters so as to develop a gene transfer vector system that can deliver genes specifically expressible in the SMC of the vasculature. The development of a gene transfer vector system that can specifically direct the expression of foreign genes in SMCs would represent an important step toward developing therapeutic strategies for treatment of human vascular diseases. In particular, restenosis of the vascular wall, which is a major complication following angioplasty to eliminate vascular occlusions during atherosclerosis.
The SM22.alpha. promoter isolated in the present invention is active only in vascular SMC of large arteries and immediate branches, i.e., no activity is seen in SMCs of veins, small arteries and visceral organs. The absence of visceral SMC activity indicates that a separable control element(s) governs SM22.alpha. in these tissues, thus making the promoter of the present invention well-suited for gene therapy of the arterial vessel wall, i.e., no leaky expression would be expected in such tissues as veins, small arteries, stomach, intestine, lung and bladder. The SM22.alpha. promoter is the only arterial SMC-specific gene control region identified to date. It therefore, represents a powerful means of directing the selective expression of foreign genes within SMCs in the vasculature, which can be delivered by gene transfer vectors.